Mushrooms belonging to the Pleurotus genus, more known as oyster mushrooms, alluding to its appearance, have brought great interest due to the versatility of their crop, which contains a wide substrate variety, including agroindustrial waste. However, studies made around its chemical composition have been left aside and are focused in evaluating the optimization of processes involving biomass production. Sajor-cajú species is an edible mushroom of clear colors, saprophyte, primary degrader and wood decomposer from which reports have not been found besides the isolation and quantification of ergosterol [IV]1. About our studies, we present in this article information related to triterpenic composition and the fatty acids identified, and as in Trigos A, we found ergosterol IV accompanied by nine additional triterpenes identified as Ergosta-2,5,7,9(ll),22-pentaene I, Ergosta-5,7,9(ll),22-tetraen-ß-ol II, Ergosta-5α,8α-epidioxy-6,22-dien-ß-ol III, Ergosta-5,7,22-trien-ß-ol (Ergosterol) IV, Ergosta-7,22-dien-ß-ol V, 19-Norergosta-5,7,9,22,tetraen-ß-ol VI, Ergosta-7-en-ß-ol VII, Ergosta-3ß-5α,6ß-trihydroxy-7,22-diene VIII, Ergosta-4,6,8(14),22-tetraen-3-one IX and Ergosta-4,6,15(16),22-tetraen-3-one X.

EXPERIMENTAL PART

The mushroom was cultivated over coffee pulp following methodology developed in the Coffee Investigations National Center CENICAFE (Chinchiná Municipality, Caldas-Colombia), using as a total growth criterion the feature of having in the border of the pileus a slight inclination towards the inside. The mushrooms were detached from the substrate, cut in little pieces, dry in oven by convection at 40 °C and grinded. 164 g of the grinded material were submitted to extraction with methanol and daily remotion of the solvent concentrating the extract under reduced pressure (53 g) to be further extracted at pH 3 with ethyl acetate and finally 12 g were obtained. The components of the extract were separated by CC in silica gel and using a mixture of toluene and ethyl acetate as eluent starting from toluene only to a ratio of 4:6. The triterpenic and particularly steroidal fraction was characterized by GC-MS using a Hewlett Packard 6890 equipment (capillary column HP5, length 30 m, inner diameter 0,33 mm and thickness 25 urn, helium as carrier gas, 4,5 at 1 mL/min from 90 °C to 300 °C at 5 °C/min), coupled to a mass spectrometer 5973 with ion source of 70 eV.

RESULTS AND ANALYSIS

The chromatographic profile of the triterpenic fraction evidenced the presence of 13 compounds; by the cleaving pattern of their mass spectra, three fatty acids commonly distributed inside the fungi kingdom were identified among these compounds: palmitic, linoleic and stearic. As diagnostic peaks, among others, were observed at m/z 60 the peak corresponding to Mc-Lafferty rearrangement, m/z 73 (base peak) of (CH2)COOH+ ion and at m/z 29, 43, 57, 71, 85 the peaks produced by the consecutive losses of 14 amu; their identity was confirmed by comparison with spectra library 2. The resting compounds (I-X) present the characteristic template of tetracyclic triterpenes, mostly sterols, with structural characteristics that vary in number and position of the unsaturations in the rings and in the side chains, and in the keto and hydroxyl groups located in different positions of the triterpenic template.

Compound I. The mass spectrum presents molecular ion at m/z 376 in accordance with the condensed formula C28H40. Peaks appear at m/z 361 (M+- methyl), 251 (M+- side chain), 236 (M+- side chain - methyl - H), 225 (M+- fission of D ring in C13 - C17 and C15 - C16), 209 (fission of D ring), 194 (209 - methyl), 156 (fission of C ring) and 141 (156 - methyl). The absence of peaks involving losses of water in their genesis, lead to discard the option of the presence of -OH groups inside the structure of the molecule. Their fragmentations agree with the ones expected for the triterpene Ergosta-2,5,7,9(1 l),22-pentaene, already isolated in the species 3.

Compound III. This compound presents a MS with a molecular ion of m/z = 428 agreeing with the condensed formula C28H4403. Inside the fragmentation pattern m/z 410 (M+- H20), 396 (M+- 02), being the latter characteristic of epidioxy sterols originated presumably by a Retro Diels Alder fragmentation generating a steroidal diene of the type Δ5,7 with its typical fragmentations. Peaks at m/z 253 (M+- side chain - H20 - 02), 211 (fission of D ring - H20 - 02) and ion at m/z 337 generated by an allylic cleavage after the loss of 02, typical of 3 - OH A6,7 epoxidioxysterols. Based on this features and by comparison with literature5, it was found that corresponds to Ergosta-5α,8α-epidioxy-6,22-dien-ß-ol, more known as ergosteryl peroxide, of wide distribution in the kingdom, but still not reported inside Pleurotus genus.

Compound IV. The compound has a molecular weight of 396, according to the mass spectrum and agreeing with condensed formula C28H440. The fragmentation pattern for this molecule is typical of sterols Δ5,7 with ions at m/z 381 (M+- CH3), 378 (M+- H20), 363 (M+- methyl - H20), 271 (M+- side chain), 253 (M+- side chain - H20), 211 (fission of D ring - H20), 143 (M+- H20 -fission C ring - methyl), 337 (M+ - 59) and 157 being the last two typical for the location of an unsaturation in C22 of the side chain; agreeing completely with the fragmentation reported in literature4 for the compound Ergosta-5,7,22-trien-ß-ol (Ergosterol), commonly distributed inside fungi kingdom.

Compound VI. The appearance of amolecular ion at m/z 380 consistent with the condensed formula C27H40O is observed. The characteristic fragmentation pattern of sterols is showed as the one observed for the compounds analyzed above, with the same unsaturated side chain as in C-22 of V (Scheme 1).

At m/z 347, a low intensity peak appears, corresponding to the loss of CH3 and H20, that for a Δ 5,7,9(11) would have a greater intensity, as in the case of ergosterol (I), where this peak corresponds to the base peak, due to the stability of the formed species, where the positive charge, product of the loss of CH3 - 19, is stabilized by conjugation with the double bonds in C-5 and C-7, and that would lead to propose the absence of the methyl group in C-19 and the presence of an aromatic ring, i.e, a 19-norsterol identified as 19-Norergosta-5,7,9,22,tetraen-ß-ol or Neoergosterol7. According to reported on literature, studies have been already made about the possible biosynthetic pathway of this kind of compounds from structurally similar sterols as ergosterol8.

Compound VIII. In the mass spectrum appear peaks which correspond to the typical fragmentations of sterols with a molecular ion at m/z 412. In the GC of the terpenoid fraction, the peak corresponding to this compound presents the largest retention time, taking to raise the possibility of having a polyhydroxylated sterol that underwent dehydration and that the peak at m/z 412 corresponds to M+- H20 resulting in a real molecular weight of 430 amu whose fragmentations agree with the peaks observed in the MS, as they are: 394 (M+- 2H20), 379 (M+- 2H20 - CH3), 287 (M+- side chain - H20), 269 (M+- side chain - 2H20) y 251 (M+- side chain - 3H20) (Scheme 2).

It was determined that this fragmentation pattern is the one corresponding to the compound Ergosta-3ß-5α,6ß-trihydroxy-7,22-diene (Cerevisterol), that has been already isolated inside the Pleurotus genus, specifically in P.ostreatus9.

Compounds IX and X. The mass spectra of these compounds allowed to establish that the substances have the same molecular ion at m/z 392 consistent with a condensed formula C28H40O and a fragmentation pattern typical of ergostan template. In their mass spectra are commonly observed losses of CH3 (m/z = 377) and the absence of peaks involving in their genesis losses of water, thus, it is possible that in carbon 3 is a carbonyl group.

Being the base peaks m/z 267 and 268 respectively, there are peaks appearing due to the loss of the side chain with composition C9H17. The lamu difference between them, is because the migration of one H of D ring favored by the presence of a double bond in C15. This hypothesis is confirmed by the analysis of fragmentations typical of this compound identified as Ergosta-4,6,15(16),22-tetraen-3-one. The substance IX corresponds to Ergosta-4,6,8(14),22-tetraen-3-one, which presents a fragmentation pattern similar to X confirmed by comparison to reports from literature 10 and specially because of peak at m/z 240 generated by fission of D ring, confirming the position of the double bond between C8 y C12 (Scheme 3).

Compounds III, V, VI, VII, IX and X have not been isolated inside the Pleurotus genus and therefore this is the first time that their isolation from sajor-cajú species is reported.

ACKNOWLEDGEMENTS

Authors thank to Chemistry Department and the Division of Research of Bogotá of National University of Colombia.